Fungi associated with the red-haired bark beetle, Hylurgus ligniperda (Coleoptera: Curculionidae) in the forest-steppe zone in eastern Ukraine

The aim of this study was to investigate the composition of the fungal community associated with the red-haired bark beetle (Hylurgus ligniperda Fabricius) in two plantations of Pinus sylvestris L. located in the Kharkiv and Luhansk regions (ca. 250 km apart) in the forest-steppe zone in eastern Ukraine. In each plantation, 48 beetles were collected from butts of living trees and 48 beetles from stems of fallen trees, i.e., a total of 96. Half of the beetles from each site were used for culturing fungi and the other half for direct sequencing the internal transcribed spacer of fungal ribosomal Rna (iTs rRna). Thirty distinct fungal taxa were identified by culturing and 31 by direct sequencing. When pooled, there were 40 fungal taxa among which Ophiostoma piceae (Münch) Sydow & P. Sydow (10.3%), Alternaria alternata (Fries) Keissler (9.7%), Ogataea neopini Nagatsuka, S. Saito & Sugiyama (8.0%), Botryotinia fuckeliana (de Bary) Whetzel (5.1%), Cladosporium sp. Link (5.1%) and Sydowia polyspora (Brefeld & Tavel) E. Müller (4.6%) were the most common. Species of the genus Ophiostoma were the most abundant and included five different taxa O. piceae, O. bicolor R.W. Davidson & D.E. Wells, O. ips (Rumbold) Nannfeldt, O. canum (Münch) Sydow & P. Sydow and O. rectangulosporium Ohtaka, Masuya & Yamaoka, all of which are known to be at most weak pathogens of trees. The plant pathogen Botryotinia fuckeliana and insect pathogens Isaria farinose (Holmskjold) Fries and Beauveria bassiana (Balsamo-Crivelli) vuillemin were also detected. Basidiomycetes were rare, among which three wood-decaying fungi Bjerkandera adusta (Willdenow) P. Karsten, Fomitopsis pinicola (Swartz) P. Karsten and Heterobasidion annosum (Fries) Brefeld were detected. In conclusion, in the forest-steppe zone in eastern Ukraine H. ligniperda is a vector of diverse communities of fungi the majority of which, if at all, are only weak pathogens of trees.


IntroductIon
Bark beetles belonging to the family Curculionidae include economically important forest pests.They are known to vector ophiostomatoid fungi (Ascomycota), including species from two phylogenetically unrelated orders, Microascales and Ophiostomatales (Seifert et al., 2013), which may cause tree diseases and discoloration of wood (Kirisits, 2004).One of these is the well-known Ips typographus L. with associated fungus and primary invader, Ceratocystis polonica (Siemaszko) C. Moreau, which can be an aggressive pathogen (Persson et al., 2009).Among others are the Dutch elm disease pathogens, Ophiostoma ulmi (Buisman) Nannf.and Ophiostoma novo-ulmi Brasier, which over the last 100 years have destroyed billions of elm trees (Ulmus spp.) worldwide (Brasier, 1991).
In recent years, a gradual increase in diversity and abundance of different bark beetles has occurred in the foreststeppe zone in eastern Ukraine (Meshkova & Sokolova, 2007).Bark beetles from the genera Hylastes (Hylastes angustatus Herbst, Hylastes ater Paykull, Hylastes opacus Erichson), Orthotomicus and in particular Hylurgus (Hylurgus ligniperda) became common causing extensive damages to young plantations, stands and timber of Pinus sylvestris (Meshkova & Sokolova, 2007).H. ligniperda breeds in logging residues including stumps, roots and logs of pine trees (Reay & Walsh, 2001, 2002;Meshkova & Sokolova, 2007).In addition, larvae of H. ligniperda may also feed on roots and butts of healthy-looking and diseased seedlings and saplings (Dumouchel & Palisek, 2002).H. ligniperda is known to be associated with several Ophiostoma species, which in trees can cause reduced increment, crown thinning, chlorosis or even the death of the tree (Kirisits, 2004;Kim, 2010).It may also vector several Grossmania and Leptographium species, which cause root diseases (Zhou et al., 2001(Zhou et al., , 2004;;Kirisits, 2004;Reay et al., 2006;Kim, 2010;Kim et al., 2011;Linnakoski, 2011;Jankowiak & Bilański, 2013).The latter may suggest that fungi vectored by H. ligniperda can be of particular importance for forest health, yet they have never been investigated in those regions of Ukraine at the south-eastern limit of the distribution of P. sylvestris in Europe.
The aim of this study was to investigate the composition of the fungal community associated with the red-haired bark beetle (Hylurgus ligniperda) in the forest-steppe zone in eastern Ukraine.
abstract.The aim of this study was to investigate the composition of the fungal community associated with the red-haired bark beetle (Hylurgus ligniperda Fabricius) in two plantations of Pinus sylvestris L. located in the Kharkiv and Luhansk regions (ca.250 km apart) in the forest-steppe zone in eastern Ukraine.In each plantation, 48 beetles were collected from butts of living trees and 48 beetles from stems of fallen trees, i.e., a total of 96.Half of the beetles from each site were used for culturing fungi and the other half for direct sequencing the internal transcribed spacer of fungal ribosomal Rna (iTs rRna)., all of which are known to be at most weak pathogens of trees.The plant pathogen Botryotinia fuckeliana and insect pathogens Isaria farinose (Holmskjold) Fries and Beauveria bassiana (Balsamo-Crivelli) vuillemin were also detected.Basidiomycetes were rare, among which three wood-decaying fungi Bjerkandera adusta (Willdenow) P. Karsten, Fomitopsis pinicola (Swartz) P. Karsten and Heterobasidion annosum (Fries) Brefeld were detected.In conclusion, in the forest-steppe zone in eastern Ukraine H. ligniperda is a vector of diverse communities of fungi the majority of which, if at all, are only weak pathogens of trees.
Multiple-banded PCR products were separated on 2.0% agarose gels, individual bands were excised and re-amplified using universal primers ITS1 and ITS4 (White et al., 1990).Resulting single-banded products were sequenced in both directions using the same primers as for PCR amplification.

statistical analyses
Richness of fungal taxa detected in beetles from different tree parts (butts and stems), different sites (Kharkiv and Luhansk) and by different methods (culturing and direct sequencing) was compared using chi-squared tests (Mead & Curnow, 1983).The relative abundance of fungal taxa was calculated from actual numbers of observations (presence/absence data) as the percentage of observations (isolates/sequences) for the total fungal community.Shannon diversity indices and quantitative Sorensen similarity indices were used to characterise the diversity and composition of fungal communities (Shannon, 1948;Magurran, 1988).

results
Of the 48 beetles of H. ligniperda used for fungal culturing, 43 (89.5%)yielded fungal growths and between one and six different fungal cultures per beetle or 77 cultures (1.8 on average) in total.Sequencing of representative cultures revealed the presence of 30 distinct fungal taxa of which 18 (60.0%)could be identified to taxon level, 8 (26.7%) to genus level and 4 (13.3%)remained unidentified (Table 1).successful amplification were obtained for all the 48 beetles used for direct amplification and sequencing of fungal ITS rRNA, with from one to three amplicons, or an average of 2.0 amplicons per beetle.Separation and sequencing of individual amplicons resulted in 98 high-quality sequences representing 31 distinct fungal taxa among which 17 (54.8%)were identified to taxon level, 9 (29.1%) to genus level and 5 (16.1%) remained unidentified (Table 1). in total, 40 fungal taxa were detected using culturing and direct sequencing.Among these, 9 (22.5%) were exclusively detected by culturing, 10 (25.0%) by direct sequencing and 21 (52.5%) by both methods (Tables 1). a chi-square test revealed no significant difference in richness of fungal taxa detected by the two methods (p > 0.05).Sorensen index of similarity of fungal communities was high (0.69) when compared between culturing and direct sequencing.In the pooled dataset, there were 32 different taxa from Kharkiv and 36 from Luhansk (Table 1), and a chi-square test revealed no significant difference in richness of fungal taxa between these sites (p > 0.05).Twenty-eight (70.0%) taxa were common to both sites.As a result, Sorensen index of similarity of fungal communities was very high (0.82) between these sites.Associated with H. ligniperda collected on different parts of trees, there were 36 fungal taxa associated with those from butts and 14 from stems, when the data from both Kharkiv and Luhansk were analyzed together (Table 1).As a result, the chi-square test revealed that the richness of fungal taxa was significantly higher for beetles from butts than from stems (p < 0.0001).Ten (25%) fungal taxa were common, resulting in a moderate (0.40) value of the Sorensen index of similarity of fungal communities associated with beetles from butts and stems.In different datasets (e.g.Kharkiv culturing butts, Kharkiv culturing stems, etc.), Shannon di-Ukraine.The distance between these sites was ca.250 km.Stands at both sites were ca.50 year-old plantations of P. sylvestris with small admixture of Betula pendula Roth and Alnus glutinosa L. Sampling of H. ligniperda was carried out in the beginning of October 2010.At each site, eight adults of H. ligniperda were collected from butts of three randomly selected living trees attacked by the bark beetles and eight from stems of three randomly selected fallen trees, resulting in a total of 48 individuals sampled at each site or 96 altogether.Living trees were defoliated and weakened trees resulting from extensive damage caused by pine sawflies in the same season.The fallen trees were harvested trees left on site that were felled during clear-felling in mid May 2010 (ca.five month before sampling the beetles).at the time of sampling, the fallen trees were dead and their wood was extensively colonized by different insects.On living trees, the bark beetle entry holes were most common in the butts (between ca.0 and 50 cm from the base), whereas in fallen trees they occurred higher up the stems (between ca.50 and 100 cm from the base).Beetles were collected from both these areas of the trunks of these trees.In both standing living trees and fallen dead trees, two 50 cm-long sections of the bark with bark beetle entry holes were carefully removed from each tree and adults of H. ligniperda were collected.The time of sampling coincided with emergence of H. ligniperda beetles from their galleries and their flying period.H. ligniperda beetles were sampled using sterilized forceps, individually placed in sterile 1.5 ml centrifugation tubes, labelled and transported to a laboratory.No visible symptoms of blue-stain fungi were noted in the sapwood at the time of sampling.Half of the beetles from each site (Kharkiv and Luhansk) and each part of a tree sampled (butt and stem) were stored at 4°C for fungal culturing and the other half at -20°C for DNA analysis.

Fungal culturing and molecular identification of taxa
One to seven days after collection, 24 beetles from each site or 48 in total were placed separately without rinsing in Petri dishes containing ca. 30 ml of Hagem agar medium and incubated at room temperature (ca.21°C) in the dark (Persson et al., 2009).Petri dishes were checked daily and outgrowing fungal mycelia were sub-cultured to new media.Fungal cultures were divided into groups based on their morphology and for species identification representative cultures from each group were subjected to sequencing of the internal transcribed spacer of fungal ribosomal Rna (iTs rRna).isolation of Dna, amplification and sequencing followed methods described by Menkis et al. (2006).Amplification by PCR was done using two primers -iTs1F (Gardes & Bruns, 1993) and ITS4 (White et al., 1990).Sequencing was carried out by Macrogen Inc., Korea.Sequences were analyzed in Seqman (version 5.07, DNASTAR, Madison, WI, USA).Databases in GenBank (altschul et al., 1997) and the Department of Forest Mycology and Plant Pathology, Swedish University of Agricultural Sciences were used to determine the identity of iTs rRna sequences.The criteria used for identification were: sequence coverage > 80%; similarity to species level 99-100% and similarity to genus level 94-98%.direct sequencing of fungi from the beetles isolation of Dna (without rinsing of the beetles), amplification and sequencing of fungal ITS rRNA obtained directly from the beetles was carried out as described by Persson et al. (2009).amplification by PCR was done in to steps: firstly using fungal specific primers nLC2 (GaGCTGaTTCCCaaaCaaCTC) and nsa3 (aaaCTCTGTCGTGCTGGGGaTa) (Persson et al., 2009), and then in a second (nested) PCR using primers ITS1F and ITS4.If only one DNA band was present per sample following nested PCR, the PCR product was used for sequencing.versity indices ranged between 1.8 and 3.3 (Table 1).Overall fungal community was composed of 90.9% Ascomycota, 7.4% Basidiomycota and 1.7% Mucoromycotina.The most commonly detected fungi were Ophiostoma piceae (10.3%),Alternaria alternata (9.7%), Ogataea neopini (8.0%), Botryotinia fuckeliana (5.1%), Cladosporium sp.

dIscussIon
The results of the present study indicate that in the forest-steppe zone in eastern Ukraine there is a species-rich community of fungi associated with H. ligniperda (Table 1).From the methodological point of view, the use of fungal culturing and direct sequencing to a slight extent complemented each other as they both detected similar fungal communities as indicated by Sorensen index of similarity, suggesting that both methods can be successfully used to study fungi associated with H. ligniperda.Furthermore, fungal communities detected at Kharkiv and Luhansk were also very similar, indicating low site specificity.This resemblance may probably be attributed to similar climatic, edaphic and forest stand conditions present at both sites e.g.high temperatures and low humidity during the growing season, nutrient poor sandy soils, similar age and composition of forest stands and low overall forest coverage.in contrast, there was notable within-tree habitat specificity since richness of H. ligniperda-associated fungi differed significantly for beetles collected on butts vs. stems (Table 1).The observed difference may probably be explained by differences in substrate quality, i.e. dead dry wood of fallen trees vs. moist living tissues of living trees.The latter should be taken into account when sampling H. ligniperda in similar studies in the future.
In the present study, 27 genera of fungi were detected among which the genus Ophiostoma was the most abundant, with five different taxa (Table 1).Despite this richness of ophiostomatoid fungi it is lower than that recorded in similar studies in which at least ten Ophiostoma spp.are reported but the composition of fungal taxa is largely the same (Zhou et al., 2001(Zhou et al., , 2004;;Reay et al., 2006;Kim et al., 2011;Jankowiak & Bilański, 2013).among the ophiostomatoid fungi detected, O. piceae is reported to be moderately or weakly pathogenic (Kirisits, 2004).However, Krokene & Solheim (1998) report that their inoculation experiments indicate that O. piceae is not pathogenic.Similarly, little-or no-pathogenic behaviour is reported for O. bicolor, O. ips, O. canum and O. rectangulosporium (Linnakoski et al., 2012) though O. ips causes lesions on Pinus spp. in South Africa (Zhou et al., 2002) and O. bicolor small dark brown areas in the wood of Norway spruce (Solheim, 1988).In addition, O. bicolor is rarely found in association with pine-infesting bark beetles (Linnakoski et al., 2012).Thus, in the forest-steppe zone in eastern Ukraine H. ligniperda is commonly associated with many taxa of ophiostomatoid fungi all of which appear to be little or non-pathogenic.Interestingly, pathogenic fungi belonging to genus Leptographium were not detected although commonly found associated with H. ligniperda in Canada and South Africa (Zhou et al., 2001;Dumouchel & Palisek, 2002).It is possible that in the regions of Ukraine studied the extreme temperatures (up to 50°C) throughout the growing season in 2010 and low levels of precipitation (Davydenko et al., 2013) negatively affected the abundance of Leptographium fungi.For example, the saprotrophic taxon Hymenoscyphus albidus (Roberge ex Desm.)W. Phillips, which was common and widespread in the region, currently appears to be rare (Davydenko et al., 2013).Instead, H. ligniperda is a vector of the plant pathogen Botryotinia fuckeliana (anamorph Botrytis cinerea), which cause grey mould disease in a wide range of plants (James et al., 1995) including seedlings of Pinus spp., Picea spp.and Abies spp.(Capieau, 2004).It is unknown whether B. fuckeliana uses H. ligniperda as a vector but it was recent-ly reported for the first time associated with the european spruce bark beetle (Ips typographus) in italy (Giordano et al., 2013) and lesser pine shoot beetle (Tomicus minor Hart.) in Poland (Jankowiak, 2008).

table 1 .
Relative abundance of fungal taxa cultured/directly sequenced from adults of Hylurgus ligniperda collected from butts and stems of Pinus sylvestris growing in the Kharkiv and Luhansk regions in eastern Ukraine.Number of the beetles used is given in the parentheses.